The cheA locus of Escherichia coli, which is required for chemotactic behavior, appears to synthesize two related polypeptide products by translating a single coding sequence from two different start points in the same reading frame. Complementation analyses of cheA mutants indicate that these two products may have different functional activities, both of which could be required for chemotaxis. The cheA locus represents the first case of overlapping genes known in bacteria and further study of this system should provide significant insight about the role of overlapping genes in non-viral systems. The objectives of this work are to clarify the mechanism of cheA expression and to investigate the functional role of the two cheA products. To test the two-start model of cheA expression, a detailed physical map of the cheA locus will be constructed by a combination of restriction site analysis and heteroduplex studies of specialized transducing phages. A restriction fragment containing the cheA coding sequence will be cloned into the single-stranded phage M13 to determine the nucleotide sequence of the promoter-proximal portion of the cheA locus. New mutations that affect cheA expression or function will be isolated with the aid of operon or protein fusions involving the cheA region and the lacZ locus, the structural gene for beta-galactosidase. Mutations that alter the relative expression or activity of the cheA products will be examined in an in vitro synthesizing system and the nature of the mutational lesion will be determined by DNA sequencing. These studies should provide useful new information about the details of cheA expression and the functional importance of the two cheA products.